Tian Ai-Ling, Elsheikha Hany M, Zhou Dong-Hui, Wu Yao-Dong, Chen Mu-Xin, Wang Meng, Chen Dan, Zhang Xi-Chen, Zhu Xing-Quan
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China.
Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Vet Parasitol. 2018 Jul 15;258:24-29. doi: 10.1016/j.vetpar.2018.06.004. Epub 2018 Jun 5.
The development of a method to rapidly diagnose Neospora caninum infection is highly desirable. Recombinase polymerase amplification (RPA), combined with lateral flow (LF) strips, is a novel approach to rapidly amplify and visualize DNA. We have developed a prototype LF-RPA assay, using primers and a probe that targeted a specific sequence in the N. caninum NC-5 gene. The N. caninum-specific LF-RPA assay was first tested on purified DNA from oocysts and amplified N. caninum DNA to detectable levels in 10 min, at a constant temperature and without the need for an expensive thermocycler. The designed RPA primers and probe displayed 100% specificity for detecting N. caninum without any cross-reaction with DNA from nine related protozoan spp. (eg Toxoplasma gondii, Sarcocystis gigantean, Sarcocystis zuoi, Hammondia hammondi, Hammondia heydorni, Eimeria cylindrica, Plasmodium falciparum, Theileria annulata and Babesia bigemina). Although, LF-RPA assay detected amounts as low as 50 fg of N. caninum DNA, it was nearly 5-fold less sensitive than previously published qPCR and nested PCR assays. We tested the diagnostic performance of the LF-RPA assay for the detection of N. caninum DNA in aborted bovine fetal tissue samples, and compared the results with those obtained from nested PCR. Out of the 75 samples examined, 18 (24%) and 17 (22.6%) tested positive using LF-RPA and nested PCR, respectively. Our results indicate that LF-RPA is a suitable assay for the rapid and reliable detection of N. caninum.
迫切需要开发一种快速诊断犬新孢子虫感染的方法。重组酶聚合酶扩增（RPA）结合侧向流动（LF）试纸条，是一种快速扩增和可视化DNA的新方法。我们开发了一种LF-RPA检测原型，使用靶向犬新孢子虫NC-5基因特定序列的引物和探针。犬新孢子虫特异性LF-RPA检测首先在来自卵囊的纯化DNA上进行测试，在恒温下10分钟内将犬新孢子虫DNA扩增到可检测水平，且无需昂贵的热循环仪。所设计的RPA引物和探针在检测犬新孢子虫时显示出100%的特异性，与来自9种相关原生动物物种（如刚地弓形虫、巨型肉孢子虫、左氏肉孢子虫、哈蒙德氏球虫、海德氏哈蒙德球虫、圆柱形艾美耳球虫、恶性疟原虫、环形泰勒虫和双芽巴贝斯虫）的DNA无任何交叉反应。尽管LF-RPA检测能检测低至50 fg的犬新孢子虫DNA，但其灵敏度比先前发表的定量PCR和巢式PCR检测低近5倍。我们测试了LF-RPA检测在流产牛胎儿组织样本中检测犬新孢子虫DNA的诊断性能，并将结果与巢式PCR的结果进行比较。在所检测的75个样本中，分别有18个（24%）和17个（22.6%）样本使用LF-RPA和巢式PCR检测呈阳性。我们的结果表明，LF-RPA是一种适用于快速可靠检测犬新孢子虫的检测方法。
